a cbx8-containing polycomb complex facilitates the transition to gene activation during es cell...

A Cbx8-Containing Polycomb Complex Facilitates theTransition to Gene Activation during ES CellDifferentiationCatherine Creppe., Anna Palau., Roberto Malinverni, Vanesa Valero, Marcus Buschbeck*

Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Badalona, Barcelona, Spain

Abstract

Polycomb proteins play an essential role in maintaining the repression of developmental genes in self-renewing embryonicstem cells. The exact mechanism allowing the derepression of polycomb target genes during cell differentiation remainsunclear. Our project aimed to identify Cbx8 binding sites in differentiating mouse embryonic stem cells. Therefore, we useda genome-wide chromatin immunoprecipitation of endogenous Cbx8 coupled to direct massive parallel sequencing (ChIP-Seq). Our analysis identified 171 high confidence peaks. By crossing our data with previously published microarray analysis,we show that several differentiation genes transiently recruit Cbx8 during their early activation. Depletion of Cbx8 partiallyimpairs the transcriptional activation of these genes. Both interaction analysis, as well as chromatin immunoprecipitationexperiments support the idea that activating Cbx8 acts in the context of an intact PRC1 complex. Prolonged gene activationresults in eviction of PRC1 despite persisting H3K27me3 and H2A ubiquitination. The composition of PRC1 is highly modularand changes when embryonic stem cells commit to differentiation. We further demonstrate that the exchange of Cbx7 forCbx8 is required for the effective activation of differentiation genes. Taken together, our results establish a function for aCbx8-containing complex in facilitating the transition from a Polycomb-repressed chromatin state to an active state. As thisaffects several key regulatory differentiation genes this mechanism is likely to contribute to the robust execution ofdifferentiation programs.

Citation: Creppe C, Palau A, Malinverni R, Valero V, Buschbeck M (2014) A Cbx8-Containing Polycomb Complex Facilitates the Transition to Gene Activationduring ES Cell Differentiation. PLoS Genet 10(12): e1004851. doi:10.1371/journal.pgen.1004851

Editor: Ragnhild Eskeland, University of Oslo, Norway

Received March 20, 2014; Accepted October 25, 2014; Published December 11, 2014

Copyright: � 2014 Creppe et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and itsSupporting Information files except for the full ChIP-seq and Microarray data that have been deposited in the GEO database under accession number GSE54053.

Funding: CC was supported by a postdoctoral FEBS long-term fellowship (http://www.febs.org/). MB is a Ramon y Cajal fellow (http://www.mineco.gob.es/). Thiswork was supported by the MINECO grants SAF2012-39749 and RYC2010-07337 (http://www.mineco.gob.es/). The funders had no role in study design, datacollection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* Email: [email protected]

. These authors contributed equally to this work.

Introduction

First identified in Drosophila, the polycomb group of proteins

share conserved domains and play an important role in

coordinated gene repression during vertebrate and invertebrate

development [1]. The prevailing view is that PRC2 and PRC1 act

in a sequential manner. The association of the three proteins Eed,

Suz12 and Ezh2 or Ezh1 leads to the formation of the core PRC2

complex. Ezh1 and Ezh2 are histone methyl transferases that

mediate the addition of up to three methyl groups to lysine 27 of

histone H3 (H3K27me1–3). The trimethylated mark is recognized

by PRC1 complexes that further mediate ubiquitination of H2A

and gene repression [2–4]. More recently, it has been shown that

PRC1 can also be recruited to chromatin in the absence of a

functional PRC2 complex [5–8].

In contrast to PRC2, the composition of PRC1 is highly

modular and much more variable. The ubiquitin ligase that

provides the catalytic activity to the PRC1 complex can be either

Ring1a or Ring1b. The complex additionally includes one of six

Pcgf proteins; one of three orthologs of polyhomeiotic and five

mutually exclusive Cbx proteins can occupy the position of the

Drosophila Polycomb protein. Cbx proteins differ in some of their

domains suggesting that they could convey different functional and

regulatory properties to PRC1 [9]. In addition a variant complex

in which RYBP replaces Cbx proteins has been shown to mediate

repression independent of the methylation status of H3K27 [7].

Mouse embryonic stem (ES) cells are characterized by their

ability to self-renew and their potential to differentiate into any of

the three germ layers. PRC maintain the pluripotency of the cells

by maintaining the developmental regulators repressed [10–12].

On differentiation ES cells acquire cell-type specific gene

expression patterns that strongly depend on the genome-wide

redistribution of the Polycomb proteins [12].

Activation of tissue specific genes correlates with the displace-

ment of Polycomb proteins and a decrease of the H3K27me3

mark during retinoic acid induced neuronal differentiation [13].

However, it has been recently shown that Polycomb proteins can

also be recruited to activated genes to attenuate the retinoic acid

associated transcriptional activation of specific genes [14]. The

important function of Polycomb complexes in the epigenetic

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changes induced by retinoic acid in mouse embryonic stem cells

has been recently reviewed by Gudas [15].

The composition of the PRC1 complex changes during the

differentiation of ES cells. Cbx7 is the primarily expressed

Polycomb ortholog in ES cells but it is quickly downregulated

during differentiation while Cbx2, Cbx4 and Cbx8 are induced

[16,17]. These studies showed that the integrity of Cbx7 was

required for stable ES cell maintenance, while Cbx2 and Cbx4

were required for balanced lineage specification. It is worth noting

that similar results have been obtained for hematopoietic stem cells

[18].

However, some important questions about Polycomb proteins

remain unanswered. Despite their overt relevance for ES cell

differentiation, it is poorly understood how Polycomb repressed

states are established and resolved. How PRCs are initially

recruited to target genes is a matter of continuous debate

(discussed in [1]). Similarly it is unclear how the transition from

a PRC repressed state to an active state is achieved. How changes

in PRC composition relate to these transitions has not been

investigated. Here, we analyzed the genome wide recruitment of

Cbx8 in ES cells induced to differentiate. We provide compelling

evidence suggesting that Cbx8 is part of a transitory PRC1

complex facilitating the activation of Cbx7-PRC1-repressed genes

during the commitment to differentiation.

Results

During ES cell differentiation Cbx8 is recruited toactivated developmental genes

We used retinoic acid (RA) to induce mouse E14 ES cells to

start differentiating towards the neuronal lineage. We confirmed

previous results [16,17] showing that Cbx8 was virtually absent in

self-renewing ES cells but potently induced on protein and RNA

levels after three days of RA-induced differentiation (Fig. 1A and

S1A Figure). To assess the genome wide distribution of Cbx8 in

differentiating ES cells, we enriched Cbx8-bound chromatin by

chromatin immunoprecipitation (ChIP) using an antibody gener-

ated against the unique part of the protein (S1B-G Figure) and

analyzed the co-precipitated DNA by direct massive parallel

sequencing (ChIP-seq). Taking advantage of the fact that Cbx8 is

virtually absent in untreated, self-renewing ES cells (Fig. 1A), we

decided to use both IgG as well as Cbx8 ChIPs from untreated

cells as negative controls. We were able to uniquely map 9–16

million reads per sample (S2A Figure). For our further analysis we

used a set of high confidence binding sites that were identified by

the overlap of peaks that were called by MACS comparing Cbx8

ChIP from RA-treated ES cells to IgG and those called comparing

Cbx8 ChIP from RA-treated ES cells to the antibody-specific

background ChIPed from untreated cells (S2B Figure). By this

method we were able to identify a subset of 171 peaks

corresponding to Cbx8 binding sites of high confidence (S1

Table). Peaks were annotated to the nearest gene if the center of

the peak was inside a window flanking the transcribed region by 3

kb. Plotting the average read coverage on genes with annotated

Cbx8 indicated that Cbx8 tends to accumulate on gene bodies but

also to spread into upstream and downstream regions (Fig. 1C).

Using this annotation we found that the large majority (141/171)

of identified peaks are associated to annotated genes (Fig. 1D). We

performed a microarray analysis comparing self renewing

untreated ES cells and differentiating cells after 3 days of RA

treatment and crossed the data with our ChIP-seq to identify

possible transcriptional changes on Cbx8 target genes. Taking into

consideration the established function of Polycomb proteins in

gene repression, we expected target genes to either not change

because they are maintained in a repressed state or to be

downregulated. We could extract data for 121 of the 141 gene-

associated peaks. We found that about one third of these Cbx8

binding sites (53 peaks) annotated to genes that displayed a more

than 1.5-fold change in gene expression. To our surprise the large

majority of these genes (44/53) was not down- but upregulated

(Fig. 1E). Most of these upregulated genes were repressed in

untreated cells as indicated by very low average probe intensities

(Fig. 1F). Though a previous report has already shown that Cbx8

can be found on a handful of activated genes in differentiating cells

[19], our data suggested that this could actually be true for a

substantial fraction of Cbx8 target genes. In order to confirm this,

we selected a panel of target and control genes and simultaneously

analyzed Cbx8 recruitment and the corresponding mRNA levels.

We were able to confirm differentiation-induced recruitment of

Cbx8 on all target genes tested while control genes were negative

(Fig. 2A). Target genes included many important key differenti-

ation genes such as Sox9, Gata6 and Nkx6-1 whose expression was

potently induced (Fig. 2B). In order to exclude the possibility that

Cbx8 binding and active transcription might occur on different

and exclusive alleles within the cell population, we analyzed the

co-occurrence of Cbx8 and H3K36me3, which is a mark of active

transcription [20], by coupled ChIP in differentiating ES cells

treated for three days with RA. First, we have analyzed five Cbx8

target genes and four non-target genes that included Oct4, Nanog,

Gapdh and Rpo. Despite the fact that Nanog is downregulated

after three days of RA treatment (Fig. 2B) it still retained some

H3K36me3 that was in a similar range as on the activated gene

Sox9 or the constitutively active gene Gapdh (Fig. 2C, left panel)

suggesting that removal of the active mark H3K36me3 follows a

slower dynamic than the actual gene repression. Taking advantage

of the fact that with Gapdh, Nanog, and Sox9 we had identified

target and non-target genes of Cbx8 with comparable H3K36me3

levels, we have used anti-Cbx8 antibody-enriched chromatin as

input material for a secondary ChIP with IgG and anti-

H3K36me3 antibody. As shown in the right panel of Fig. 2C,

we found a clear enrichment of H3K36me3 over IgG on Sox9 and

on the other Cbx8 target genes but not on non-target genes such

as Gapdh or Nanog. As chromatin ranging in size between 300–

500 bps has been used for these experiments H3K36me3 and

binding of Cbx8 could be occurring on different H3 tails on the

Author Summary

Cell fate transitions have long been known to beaccompanied by alterations in chromatin structure. Butonly during the last few years has it become clear thatchromatin modifications form the molecular basis of anepigenetic memory that defines cell identity. The Poly-comb Group Proteins (PcGs) form two major proteincomplexes known as polycomb repressive complexes 1and 2 (PRC1 and PRC2). Their function is essential for themaintenance of transcriptional repression during embryo-genesis through the methylation of the lysine 27 onhistone H3 and the subsequent ubiquitination of histoneH2A. The chromobox homolog 8, Cbx8, which is part ofthe PRC1 complex, is therefore generally defined as arepressor of gene transcription. The genome wide profilingof Cbx8 during the early steps of mouse embryonic stem(mES) cells differentiation provided us with surprisingresults involving Cbx8 in gene activation. Our results pointout that Cbx8 is part of a PRC1 complex involved in thetransition from a Polycomb repressed state to an activestate.

Cbx8 Facilitates Gene Activation


same or even neighboring nucleosomes. The observation that

Cbx8 can be simultaneously detected on the same locus provides

further support to the idea that Cbx8 is recruited to genes that

become actively transcribed.

Cbx8 facilitates gene activationTo study the functional importance of Cbx8 for the activation of

differentiation genes, we used lentiviral vectors expressing two

different short hairpin RNAs (shRNA) directed specifically against

the Cbx8 transcript. After selection stably transduced cells were

used for the study. Both shRNAs efficiently repressed the

expression of Cbx8 on both the mRNA and protein levels in ES

cells treated with RA (Fig. 3A,B). The activation of upregulated

Cbx8 target genes was significantly decreased in cells depleted for

Cbx8 (Fig. 3C). Among the Cbx8-sensitive genes were several

pivotal regulators of differentiation processes such as Sox9 and

Nkx6-1, that have been shown to be important transcription

factors required for normal brain development [21,22]. The

reduction of Cbx8 occupancy was similarly efficient on up- as well

as downregulated genes (Fig. 3D), however, the reduction in Cbx8

levels didn’t affect the repression of its target genes Prdm14 and

Otx2 (Fig. 3C). Importantly, the non-target genes Oct4 and

Nanog, which encode the regulators of pluripotency, were

similarly repressed in Cbx8 deficient and control cells (Fig. 3C,D).

Plotting enriched gene ontologies according to their similarity in a

semantic space illustrates a clear overrepresentation of both

transcriptional and differentiation regulators (Fig. 4A), which are

the classical categories of Polycomb target genes in self-renewing

ES cells [12]. Gene ontologies related to neuronal development

were preferentially enriched in the subgroup of activated Cbx8

Fig. 1. Cbx8 is upregulated and recruited to activated genes during ES cell differentiation. (A) Lysates from E14 mouse embryonic stem(mES) cells treated or not with 1 mM retinoic acid (RA) during 3 days were analyzed by Western blotting using anti-Cbx8 and anti-Cbx7 antibodies.Histone H3 was used as loading control. (B) ChIP-seq profiles of IgG and Cbx8 of selected genes in ES cells treated with RA for 3 days or left untreatedare shown using the UCSC Genome Browser. (C) Normalized read distribution of genes with associated Cbx8 peaks in ES cells treated with RA forthree days. Please note that average read distribution on gene bodies is plotted after normalization for the size of transcribed regions, while regionsupstream of the transcription start site (TSS) and downstream of the transcription end site (TES) are plotted linear. (D) The large majority of Cbx8peaks are associated with genes considering a window of the transcribed region 63 kb. (E) For 121 of the 141 gene-associated peaks we extractedtranscriptional information from whole genome expression microarrays of ES cells treated with RA for 3 days and untreated control cells. Theproportion of genes with upregulated, downregulated or unchanged expression using a 1.5-fold change as cut-off is plotted in a pie chart andabsolute numbers are indicated in brackets. (F) Average probe intensities corresponding to the up- and downregulated Cbx8-target genes indicatedin E are shown in a boxplot for untreated and RA-treated cells. Note that probe intensities from 6-7 correspond to background levels. All data plottedin D–F is detailed in S1 Table.doi:10.1371/journal.pgen.1004851.g001



genes but not those target genes that did not show any change in

gene expression (S3A Figure). Downregulated genes were not

sufficient in number to yield a result in gene ontology analysis. We

compared our genome wide Cbx8 binding profile in differentiating

ES cells with published ChIP-seq data obtained from self-renewing

ES cells [17]. The binding of Cbx8 in RA-treated differentiating

ES cells mirrors the binding of PRC1 proteins Ring1b and Cbx7

within H3K27me3 domains in untreated self-renewing ES cells

(Fig. 4B). As shown in Fig. 4C, this holds true for the vast majority

of Cbx8 binding sites in RA-treated ES cells as 133/171

overlapped with sites bound by Cbx7 in self-renewing ES cells.

Similar overlaps were observed with Ring1b and H3K27me3 (S3B

Figure).

Cbx8 acts as part of a PRC1 complexThe unexpected link between Cbx8 recruitment and gene

activation prompted us to analyze whether Cbx8 acted alone, as

part of a PRC1 or part of a different complex. First we compared

the dynamics in occupancy of Cbx8 and Ring1b, which is the least

variant component of PRC1. As already suggested by the ChIP-

seq data (Fig. 4B), Ring1b was strongly enriched on target genes in

self-renewing ES cells. While the recruitment of Cbx8 initially

increased and peaked after three days of retinoic acid induction,

the binding of Ring1b decreased progressively reaching back-

ground levels at day five of differentiation (Fig. 5A). At day five of

retinoic acid induced differentiation, the Cbx8 occupancy dropped

to very low levels similar to Ring1b. In contrast, H2A

ubiquitination, mark set by Ring1b [23], persisted over the entire

time course (Fig. 5A). In order to understand whether Cbx8 and

Ring1b are acting together, we decided to identify the proteins

that bind Cbx8 during ES cell differentiation. Therefore, we

generated ES cells stably expressing epitope-tagged Cbx8

(Fig. 5B). Exogenous Cbx8 expressed in untreated ES cells bound

to the same target genes as endogenous Cbx8 in RA-treated ES

cells suggesting that the epitope does not affect its function (S4A

Figure). We harvested cells at day three of differentiation which

corresponds to the time point with maximal recruitment of

endogenous Cbx8 to target genes (Fig. 5A). Cbx8 and interacting

proteins were enriched by affinity purification, analyzed by mass

spectrometry and significantly enriched proteins were ranked

according to their abundance (S2 Table). After the bait protein

Cbx8, the top five ranked proteins were the PRC1 subunits

Ring1a/b, Phc2 and the Pcgf proteins Mel18 and Bmi1. Three

additional PRC1 subunits were found in lower abundance

(Fig.5B). Next, we tested whether Ring1b and H2A ubiquitination

co-occur with Cbx8 on genes. Therefore, we have used anti-Cbx8

Fig. 2. Cbx8 binding coincides with gene activation. (A) The occupancy of Cbx8 on genes was analyzed by ChIP in untreated ES cells anddifferentiating cells treated with RA for 3 days. Data is plotted as percentage of ChIP-enriched DNA in respect to DNA from input material. Error barsdenote s.d., and n = 3. (B) qRT-PCR analysis of mRNA levels in the same cells as A. Levels in untreated cells have been set to one. Error bars denote s.d.,and n = 3. (C) ChIP and ReChIP analyses performed with differentiating ES cells treated with RA for three days are shown. Anti-H3K36me3 and controlIgG antibody were used in a primary ChIP (left panel) or in a secondary ChIP performed on material enriched in a primary ChIP with anti-Cbx8antibody (right panel). ReChIP data is plotted relative to IgG. Error bars denote s.d., n = 3.doi:10.1371/journal.pgen.1004851.g002

Fig. 3. Knockdown of Cbx8 reduces transcription of activated target genes. (A) The mRNA levels of Cbx8 in control cells (sh Scr) and aftershRNA-mediated knockdown of Cbx8 using lentiviral transduction of two independent hairpins (#1 and #2) were analyzed by qRT-PCR before andafter treatment with RA. Error bars denote s.d., and n = 3. (B) Western blot analysis of Cbx8 in RA-treated ES cells. Histone H3 was used as loadingcontrol. (C) Transcript expression levels of genes in cells expressing Cbx8-specific shRNAs or control shRNA (indicated by minus) were analyzed. Colorcode of target genes is according to Figs. 1F and 2B. For each gene, values are plotted relatively to control treated with RA. Error bars denote s.d.;n = 3; *, p,0.05 (comparing to RA-treated control). (D) The occupancy of Cbx8 on the same genes was analyzed by ChIP. Error bars denote s.d., andn = 3.doi:10.1371/journal.pgen.1004851.g003



antibody-enriched chromatin as input material for a secondary

ChIP with IgG, anti-Ring1b and anti-ubiquitinated H2A

antibody. As shown in Fig. 5C we could detect co-enrichment

on the target genes Sox9, Nkx6-1, Lhx2 and Gata2 but not on the

non-target genes Oct4, Rpo or Gapdh. Taken together, these

results suggested that Cbx8 acts as part of a PRC1 complex.

To further support this, we have analyzed the occupancy of

Cbx8 target genes by Ring1b, Cbx7 and H2A ubiquitination in

RA-treated cells after Cbx8 knockdown. The resulting reduction

of Cbx8 occupancy (Fig. 3D) correlated with a small but consistent

reduction of Ring1b on several Cbx8 target genes without

affecting non-target genes (Fig. 6). Notably, global Ring1b protein

levels were not affected (S4B Figure). However, we observed an

increased incorporation of Cbx7 that could partially compensate

for Cbx8 loss (Fig. 6). This was not the consequence of an

upregulation of Cbx7 expression as knockdown of Cbx8 did not

affect the mRNA levels of Cbx7 or other Cbx proteins (S4C

Figure). Moreover we found that Cbx8 loss did not affect the levels

of H2A ubiquitination on its target genes (Fig. 6). In order to

address the question whether Cbx8-containing PRC1 on activated

genes is repressive or activating, we stably interfered with the

expression of Ring1b, the least variant component of PRC1.

When analyzing Ring1b knockdown cell after 3 days of RA

treatment we did not observe any compensation by Ring1a but a

slight increase in Cbx8 mRNA (S4D Figure). Under these

conditions the Cbx8 target gene Gata2 was further upregulated

while other target genes such as Nkx6-1 and Sox17 were less

activated (S4D Figure). These results are difficult to interpret as

Fig. 4. Cbx8 target genes are PRC1 target genes in self-renewing ES cells. (A) Cbx8-enriched GO categories are visualized using REVIGO [48]which allows to cluster GO according to their similarity in a semantic space. Only GO categories with an adjusted P-value of 0.001 or less are shownusing ‘‘medium’’ for the allowed semantic similarity. (B) ChIP-seq profiles of H3K27me3, Ring1b, Cbx7 and Cbx8 in ES cells in self-renewing conditionsand of Cbx8 and IgG in RA-treated ES cells are shown using the UCSC genome browser. (C) Venn diagram showing the overlap of Cbx8 target genesin RA-treated cells with those of Cbx7 in untreated, self-renewing ES cells.doi:10.1371/journal.pgen.1004851.g004



Fig. 5. Cbx8 is part of a PRC1 complex and transiently bound to activated genes. (A) Time course of RA-induced differentiation of ES cells.mRNA levels and occupancy by Cbx8, Ring1b and H2A K117 mono-ubiquitination (H2Aub1) were analyzed by qRT-PCR and ChIP, respectively. Errorbars denote s.d.; n$3. (B) FLAG-affinity purifications from mock and FLAG epitope-tagged (e)-Cbx8 expressing cells treated with RA for 3 days,analyzed by LC-MS/MS. Enriched proteins were ranked according to abundance (see also S2 Table). Relative abundance to bait is indicated with +, 1–10%; ++, 10–30%; +++, 30–50%; and ++++, .50%. (C) ReChIPs using anti-Ring1b, anti-H2Aub1 antibodies and IgG as control were performed onmaterial enriched in a primary ChIP with anti-Cbx8 antibody. Data is plotted relative to IgG. Error bars denote the variation of the mean of twoindependent experiments.doi:10.1371/journal.pgen.1004851.g005



knockdown of Ring1b interferes with all PRC1 complexes present

prior and after RA treatment.

Cbx8-containing PRC1 binds to persisting H3K27me3We then focused our attention on the mechanism that recruits

Cbx8 to its target genes. An obvious possibility is that it binds

directly to H3K27me3. Although activated Cbx8 target genes

progressively reduced their H3K27me3 levels, in contrast to

Ring1b, this reduction occurred only very slowly and even after

five days of differentiation genes still retained half-maximal or even

higher levels of H3K27me3 (Fig. 7A). Depletion of Cbx8 did not

affect the amount of H3K27me3 detectable 3 days after RA

treatment (Fig. 7B). We then analyzed by ChIP-ReChIP if Cbx8

and H3K27me3 co-occur on target genes in ES cells treated for 3

days with RA. Indeed, we found that Cbx8 and H3K27me3 co-

existed on Cbx8 target genes (Fig. 7C). This suggests that also in

the context of gene activation the interaction of Cbx8 with

H3K27me3 is the most likely mechanism of recruitment to

chromatin.

In addition to H3K27me3, at day 3 of RA treatment we also

detected some acetylation of the same residue on Cbx8 target

genes (S5B Figure). This H3K27ac was of low level when

compared to an enhancer that was previously described to be

marked by H3K27ac in ES cells (S5A Figure) [24]. Curiously,

knockdown of Cbx8 lead to a modest but significant decrease of

H3K27ac (S5B Figure). The presence of these two mutually

Fig. 6. Loss of Cbx8 is partially compensated by Cbx7. The presence of Ring1b, Cbx7 and H2A ubiquitination (H2Aub1) in control cells (sh Scr)and after shRNA-mediated knockdown of Cbx8 using two independent hairpins was analyzed by ChIP after 3 days RA treatment. IgG was used asbackground control and is indicated by minus signs. For each Cbx8 target gene values are plotted relatively to control cells. Enrichments on-targetgenes are plotted relative to the average enrichment on all six target genes in control cells. Error bars denote s.d.; n = 3; *, p,0.05. (compared tocontrol cells).doi:10.1371/journal.pgen.1004851.g006



Fig. 7. Cbx8 binds to persisting K27 methylation during differentiation. (A) Trimethylation of lysine 27 of Histone H3 was analyzed by ChIPduring a time course of RA-induced differentiation of ES cells. Control ChIPs with IgG are shown in the top panel on the same scale. Error bars denotes.d.; n$3; n.d. = not done. (B) ChIP of H3K27me3 (+) and IgG (-). For each Cbx8 target gene values are plotted relatively to control cells. Enrichmentson-target genes are plotted relative to the average enrichment on all six target genes in control cells. Error bars denote s.d.; n = 3. (C) ReChIPs usinganti-K27me3 antibody and IgG as control were performed on material enriched in a primary ChIP with anti-Cbx8 antibody. Data is plotted relative toIgG. Error bars denote the variation of the mean of two independent experiments.doi:10.1371/journal.pgen.1004851.g007



exclusive H3K27 marks provided us with a valuable tool to

interrogate the preferential binding of Cbx8. First we titrated both

antibodies to reach similar enrichment in ChIP assays (S5C

Figure). Then we used the enriched material to perform a

sequential ChIP for Cbx8. As shown in S5C Figure, Cbx8

preferentially bound K27me3-marked chromatin.

Cbx8 is required for efficient gene activation but notsufficient to induce it

Cbx7 and Cbx8 are expressed in an almost mutually exclusive

manner in self-renewing and differentiating ES cells, respectively

[16,17]. To further gain additional insight into the functional

relevance of the switch from Cbx7 to Cbx8 on target genes, we

generated mouse embryonic stem cells that stably express

exogenous Cbx8 and analyzed them in self-renewing conditions

while cells expressing exogenous Cbx7 were analyzed in differen-

tiating cells after 3 days of retinoic acid induction (Fig. 8A). When

expressed in self-renewing cells, exogenous Cbx8 was able to

efficiently outcompete Cbx7 for its target genes (Fig. 8B). The

enforced recruitment of exogenous Cbx8 achieved under these

conditions was two-to-three-fold higher than that observed in

differentiating cells for the endogenous protein (Fig. 8B), although,

this did not affect the low expression level of these genes (Fig. 8C).

In the converse experiment during differentiation, Cbx7 overex-

pression significantly reduced the activation of Cbx8 target genes

(Fig. 8C). Although enrichment of overexpressed Cbx7 on target

genes did not reach the levels of the endogenous protein in self-

renewing cells, it resulted in an efficient displacement of Cbx8

(Fig. 8D).

Discussion

Cbx8 is part of a transition PRC1 acting during initialgene activation

Taken together our results support a model in which PRC1

containing Cbx8 replace PRC1 containing Cbx7 on developmen-

tal genes, which facilitates the transition from a repressed

chromatin state to gene activation during early ES cell differen-

tiation (Fig. 8E). Prolonged gene activation results in eviction of

PRC1 complexes despite some persisting H3K27me3 and H2A

ubiquitination. This mechanism affects several key regulatory

genes and thus probably contributes to a robust execution of

differentiation programs.

Our data supports the idea that Cbx8 acts in the context of an

intact PRC1 complex. Initially, we considered also two other

possibilities: Cbx8 could act as monomer competing with

repressive PRC1 complexes for H3K27me3 binding sites, or

Cbx8 acts in complex with non-polycomb proteins that are

activating. Indeed, in leukemia cells Cbx8 has already been

described as a component of activating complexes containing

Tip60 and MLL-AF9 [25]. However, performing mass spectro-

metric analysis of affinity purified Cbx8-complexes from differen-

tiating ES cells we could not detect any of these proteins, but

rather found other PRC1-components acting as main binding

proteins (Fig. 5B). Moreover, we have shown that Ring1b was

enriched on Cbx8 immunoprecipitated chromatin compared to

IgG (Fig. 5C) and that the knockdown of Cbx8 resulted in a small

but detectable reduction of Ring1b occupancy on genes (Fig. 6).

Since we would have expected the opposite if Cbx8 acted as a

monomer on target genes, this observation taken together with our

co-immunoprecipitation data strongly argued for Cbx8 to be

functioning in the context of a PRC1 complex.

Replacement of Cbx7-containing PRC1 for a Cbx8-containing complex

One fundamental question is how a Cbx8-containing PRC1 is

able to contribute to gene activation. A plausible explanation

could be that Cbx8-containing PRC1 is simply less repressive than

the PRC1 containing Cbx7 that it replaces. This prompted us to

test whether Cbx8 has an influence on PRC1 activity. When

monitoring the ubiquitination of H2A, we found that its levels on

Cbx8 target genes neither decreased during the first five days of

differentiation nor changed on depletion of Cbx8 (Figs. 5A, 6). It

has been shown that Ring1b mediates chromatin compaction and

gene repression independently of its catalytic activity [26]. In that

regard, it would be interesting to test whether changes in PRC1

composition affects its capacity to compact chromatin. If Cbx7-

containing PRC1 complexes induced a higher degree of chromatin

compaction, this could possibly be relaxed on replacement by

Cbx8. Knockdown of Ring1b itself let to the further activation of a

Cbx8 target gene but to a reduction in the activation of several

other Cbx8 target (S4D Figure). It is intriguing to speculate that

the outcome could depend on the ratio of Cbx7 and Cbx8-

containing PRC1 complexes present at the time point of analysis

and the gene-specific kinetics of activation. However, the

interpretation of such data is complicated by the large number

of binding sites of Ring1b as its other target genes could exert

indirect effects and the fact that Ring1b occurs in both PRC1 and

non-PRC1 complexes [27]. Finally, of course we cannot exclude

the possibility that Cbx8-containing PRC1 complexes are able to

recruit activating proteins in a very transient way, which would not

be captured by our purification and mass spectrometric analysis.

Dynamic gene regulation and slower turnover ofchromatin marks

Both the replacement of Cbx7-containing PRC1 for Cbx8

loaded complexes and the complete eviction of all PRC1 after

prolonged activation occurs in the context of persisting

H3K27me3 and H2A ubiquitination. This reiterates that these

marks have a slow turnover and are not necessary a reflection of

gene activity or repression, in particular during dynamic cell fate

transitions. On Cbx8 target genes we could detect low levels of

H3K27ac that were further reduced mildly but significantly in cells

depleted for Cbx8 (S5B Figure). Whether Cbx8-dependent

H3K27ac is a cause or consequence of enhanced transcription is

unclear. H3K27ac could positively affect chromatin accessibility

for transcription-supporting factors. However it is more likely that

the observed low level of H3K27ac is a collateral consequence of

an increased concentration of histone acetylases travelling with

Polymerase II.

PRC1 modularity during ES cell differentiationThe modularity of PRC1 is illustrated by the fact that there are

180 theoretical combinations for assembling the different PRC

subunits. The real number of different PRC1 complexes existing

under different physiological conditions is likely to be much lower

due to mutually exclusive expression patterns and preferential

binding between subunits. The systematic proteomic and epige-

nomic analysis of all six PCGF proteins shed some initial light on

the different compositions and genomic distributions of PRC1

complexes [28]. On the one hand proteomic studies allowed the

identification of new PRC1 components such as RYBP and YAF2,

while on the other hand they increased doubts about the

genuineness of subunits that had previously been considered to

be canonical; such as Cbx6. A major challenge for the field is to

understand how the modularity of PRC1 is regulated and how it



contributes to cellular functions. Self-renewing ES cells primarily

express two PRC1 complexes containing either Cbx7 or RYBP

[7]. Whereas Cbx7-PRC1 mediates early repression of differen-

tiation genes by binding to H3K27me3, RYBP-PRC1 binds

independently of the methylation status of H3K27 and is

associated with lower levels of Ring1b and H2A ubiquitination

and occupies genes that are less repressed [29]. Cbx7 and Cbx8

are expressed in an almost mutually exclusive manner in self-

renewing and differentiating ES cells, respectively. Although invitro the chromodomain of Cbx7 has higher affinity to

H3K27me3 than the one of Cbx8 [30], in cells we found that

both proteins were able to efficiently replace each other on genes.

Enforced expression of Cbx7 in differentiating cells was able to

compete with Cbx8 for its target genes and to reduce gene

activation. In the converse experiment similarly efficient replace-

ment of Cbx7 by exogenous Cbx8 in self-renewing ES cells was

not sufficient to induce derepression (Fig. 8A–D). These results

place Cbx7 over Cbx8 in the functional hierarchy of Polycomb

proteins. Future studies will have to assess in greater detail how

different PRC1 complexes regulate cell fate decisions. Our

preliminary data shows that ES cells that maintain 30–50%

reduced Cbx8 expression are qualitatively able to differentiate into

Fig. 8. The exchange of Cbx7 for Cbx8 is required but not sufficient to promote the differentiation of mES cells. (A-D) Parental ES cellstreated with RA for three days or left untreated were compared to untreated ES cells expressing exogenous Cbx8 and differentiating ES treated withRA for three days and expressing exogenous Cbx7. Consistent color coding of cells and treatments is as indicated in A. (A) The expression levels ofendogenous and exogenous epitope (e)-tagged Cbx proteins is shown by Western blot of lysates from stable transfected ES cells (e-Cbx7 and e-Cbx8,respectively) and parental control cells. Exogenous and endogenous protein bands are marked by one or two asterisks, respectively. Cells weretreated with RA for three days as indicated. Histone H3 was used as loading control. (B) The occupancy of Cbx7 and Cbx8 on selected genes wasanalyzed by ChIP in untreated ES cells overexpressing Cbx8 (red bars) and parental control cells treated with RA for three days (black bars) or leftuntreated (grey bars). ChIP-Input ratios for each gene and antibody are shown relative to the maximal enrichment. IgG is shown relative to Cbx8 ChIP.Error bars denote s.d.; n = 3; * = p,0.05. (C) Relative mRNA levels of the same genes as in B measured by qRT-PCR are shown. Error bars denote s.d.;n = 3; * = p,0.05; n.s. = not significant. (D) ChIP of Cbx8 or Cbx7 in Cbx7-overexpressing cells treated with RA for three days (blue bars) and controlcells treated with RA (black bars) or left untreated (grey bars). ChIP-Input ratios for each gene and antibody are shown relative to the maximalenrichment. IgG is shown relative to Cbx8 ChIP. Error bars denote s.d.; n = 3; * = p,0.05. (E) Cartoon illustrating our finding that Cbx8-containing PRC1complexes replace Cbx7-containing PRC1 complexes during the initial activation of differentiation genes. During a metastable transition statebinding of Cbx8-containing PRC1 co-occurs with the presence of both repressive marks such as H3K27me3 and active marks such as H3K36me3.Prolonged activation results in loss of Cbx8-containing PRC1 that precedes the removal of H3K27me3.doi:10.1371/journal.pgen.1004851.g008



Tuj1-positive neurons with neurite outgrowth following 11 days of

a long-term differentiation protocol (adapted from [31]), but seem

to do so in a less efficient way (S6 Figure).

Polycomb proteins and gene activationThe number of observations linking Polycomb proteins and

active gene transcription are increasing. Here we report that the

transient recruitment of PRC1 containing Cbx8 facilitates the

transition from a Polycomb-repressed to a fully active state of key

regulatory genes during early differentiation. Others have

suggested that binding of Cbx8 to active genes could mark these

for later repression [19]. Our data does not support this as we find

Cbx8 recruitment to be only transient and all PRC1 to be entirely

evicted after prolonged gene activation (Fig. 5).

It is worth to point out that Cbx8 target genes that got repressed

after treatment with retinoic acid were not affected by the

knockdown of Cbx8. This can be explained by a possible

compensation by other canonical repressing PRC1 complexes or

a recent finding showing that Polycomb protein recruitment is

rather a consequence than a cause of initial gene silencing [32].

Studying differentiating myocytes, others have reported that

Ezh1 associates with actively transcribed genes and further argued

for a positive function in which Ezh1 could be required for the

recruitment of Polymerase II [33]. In contrast, Pombo and

colleagues suggested that their observed Polycomb-binding to

transcribed metabolic genes was the consequence of a continuous

switching between an active and a Polycomb-repressed state

restraining transcriptional elongation [34]. In Drosophila cells,

PRC1 was found to indirectly associate with some active as well as

inactive genes by binding to structural cohesin proteins [35]. The

authors argued again for a more active role on active genes by

suggesting that PRC1 could be required for allowing the

phosphorylation that makes Polymerase II elongation competent.

A large body of additional work is needed to sort out the relation

between Polycomb-bound and transcribed chromatin states in

greater detail. In this endeavor it will be important to carefully

distinguish between passive contributions from reductions in

repressive potential and genuine contributions to transcription

initiation and elongation.

Materials and Methods

Antibodies and plasmidsWe produced a specific polyclonal antibody against mouse

Cbx8 by immunizing rabbits with a His-tagged fragment of Cbx8

protein encompassing amino acids 201–360. Serum was pre-

cleared with sepharose and passed over a column containing a

fusion protein of glutathione-S-transferase (GST) and amino acids

201–360 of Cbx8 covalently cross-linked to Glutathione sephar-

ose. Anti-Cbx8 antibody was eluated with low pH, dialyzed and

stored in PBS with 20% glycerol. The antibody performed in a

similar way to antibodies previously described and kindly provided

by Kristian Helin [13]; (S1 Figure). In addition we made use of the

following antibodies: anti-IgG (Abcam), anti-H3 C-terminal

(Abcam) and anti-H3K27me3 (Millipore), anti-Flag M2 (Sigma-

Aldrich), anti-Ring1b provided by Luciano Di Croce [36], anti-

H3K27Ac and anti-Cbx7 (Abcam), anti-H3K36me3 (Abcam) and

rabbit monoclonal anti-ubiquityl-H2A (Lys119, D27C4, Cell

Signaling Technology). The amounts and concentrations used

for ChIP and western blotting is given in S3 Table. Expression

plasmids and pLKO-1 constructs for shRNA-mediated knock-

down were generated with standard PCR and cloning techniques.

For stable expression in ES cells, cDNAs were cloned in frame

with a multiple-epitope tag into a vector containing CAG

promoter and an IRES-puromycin resistance gene cassette [37].

A modified and gateway cloning-adapted (Life Technologies)

version was kindly provided by Diego Pasini.

Cell culture, gene transduction and cell differentiationE14Tg2A.4 mouse ES cells were cultured as previously

described [38]. Cells were transduced with lentiviral shRNA

cassettes essentially as described before [39]. Transduced cells

were selected with 2 mg/ml puromycin. For shRNA sequences see

S3 Table. For the generation of stably expressing ES cell clones,

the above described CAG promoter driven vectors were trans-

fected into mouse ES cells E14 using Lipofectamine 2000 (Life

Technologies). Stable transfectants were selected with 2 mg/ml

puromycin and analyzed by RT-PCR and western blot for

transgene expression. For neuronal differentiation, 1 mM all trans

retinoic acid (RA) was added directly to cells in medium without

leukemia inhibitory factor. Unless indicated otherwise in Figure

legends, cells were collected after 3 days of RA treatment.

Protein and RNA analysisLysis and western blot analyses were performed as previously

described [40]. Following the supplier’s instructions, RNA was

purified from 26106 cells using the RNeasy minikit (Qiagen), with

a DNase I digestion step to avoid any potential DNA contami-

nation. Total RNA (1 mg) was reverse transcribed using a cDNA

synthesis kit (Roche Diagnostics) and oligo(dT) primers. Relative

cDNA levels were quantified by quantitative PCR (qRT-PCR).

Values were normalized to the expression of two housekeeping

genes (Rpo and Gapdh). For gene expression analysis, four

biological replicates of RA treated (3 days) and untreated ES cells

were used for each condition and samples were prepared and

hybridized to SurePrint G3 Mouse GE 8660K Microarrays

(Agilent technologies) following the supplier’s instructions. Anal-

yses were essentially performed as described [41] selecting

differentially expressed probes with a FDR of 0.05 and fold

change of .1.5.

ChIP and ChIP-seqChromatin fragmented to a size ranging from 300–500 bps and

immunoprecipitation (ChIP) experiments were performed essen-

tially as previously described [42]. ChIP-reChIP experiment was

performed as described elsewhere [34]. The sequences of all

oligonucleotides used here are provided in the S3 Table. Unless

indicated otherwise, ChIP results are given as the percentage of

the amount of ChIP-enriched DNA relative to the amount of

DNA isolated from one tenth of input material measured by

quantitative PCR. For ChIP-sequencing (ChIP-seq), 10 ng of

DNA was enriched by ChIP and fluorimetrically quantified with

PicoGreen. Library generation and direct massive parallel

sequencing on an Illumina genome analyzer were performed

according to the supplier’s instructions. Reads obtained were

cleaned based on quality, trimmed using the ShortRead package

in R [43] and aligned with the mouse genome (NCBIM37/mm9)

using Bowtie version 0.12.7 [44], two mismatches were allowed for

the alignment within the seed, only reads mapping to a single

position in the genome were used. To detect genomic regions with

significant enrichment we used MACS software version 1.4.1 [45].

For peak calling of Cbx8 in RA-treated ES cells we used a p-value

cut-off of 161024 and a FDR of 5%. Both IgG and Cbx8 from

self-renewing cells (that do not express Cbx8) were independently

used as control libraries. Only peaks called in both cases (minimal

overlap of 50 bps) were accepted as high confidence target peaks

and further analyzed. A subset was validated by direct ChIP.

Peaks were annotated using ChIPpeakAnno package [46]. Genes



were considered to be target genes if the center of a peak was

found in the transcribed region 63 kb using the transcript set of

Mouse Ensembl Gene (based on assembly NCBIM37/mm9). In

cases where a peak annotated to two genes, the nearest gene was

selected and identified by the minimal distance between peak

center and transcribed region. Un ambiguous peak that was

found inside an intron of three differently annotated transcripts

(chr10:3310058,3311329) was excluded from the analysis. To

calculate the normalized enrichment profile we have used

ngs.plot ver 2.0 [47]. The gene ontology analysis was performed

using the function ‘‘getEnrichedGO’’ from ChippeakAnno

package, we used the Ensembl gene ID and accepted GO

categories with an adjusted P-value of 0.001 or less. P-values were

calculated using the multiple adjusted Benjamini-Hochberg

method. A value of ten was set as minimum count in the

genome for a GO term to be included. For visualizing GO

categories we used REVIGO [48], using the following param-

eters: ‘‘Medium’’ for the allowed similarity and ‘‘SimRel’’ for

semantic similarity measure. Gene ontologies were grouped and

genes were scored if they were associated to one or more ontology

terms per group.

Proteomic analysisFor proteomic analysis, 26108 differentiated ES cells expressing

Flag-epitope tagged Cbx8 and parental control cells were collected

with PBS. Nuclei were isolated using sucrose buffer and nuclear

extract was separated from chromatinic fraction by a high salt

extraction protocol followed by ultracentrifugation (1 h

50000 rpm). The soluble fraction containing nuclear extract was

diluted to isotonic concentration followed by a preclear step using

sepharose beads. Binding to Anti-Flag M2 Beads (Sigma-Aldrich)

was performed in a rotating wheel during 2 h. Then, Anti-Flag

M2 Beads were passed into a column for further enrichment and

washing steps. Beads were collected into tubes for elution with

50 mM NaHCO3 and 0, 5% SDS. Eluated proteins were

precipitated with cold acetone and frozen dry pellets were sent

to a Proteomics facility for mass spectrometry analysis. Samples

were digested with trypsin and 1 mg of each sample was injected in

an Orbitrap Velos to LC-MS/MS analysis. Data was searched

using an internal version of the search algorithm Mascot against a

SwissProt_Mouse database (July 2013). Protein identification and

peptides identified for each protein were identified using Proteome

Discoverer v1.4, which gives an approximate estimation of protein

amount with the average peak area of the 3 top peptides for a

given protein. The number of peptides identified for each protein

is a parameter of quality, the more peptides the better. Peptides

were filtered based on the 1%FDR. Only proteins enriched more

than 10 fold and with a coverage of at least 5% were considered

for analysis.

StatisticsUnless indicated otherwise, qRT-PCR and ChIP data is

represented as the mean of three independent experiments and

errors denote the standard deviation and stars indicate p-values

below 0.05 as determined by the two-tailed Student’s T-tests.

Individual ChIP experiments are normalized to the average

enrichment observed in the experiment. Means and errors of

three experiments are scaled to represent the average of all

experiments.

Accession numbersChIP-seq and Microarray data have been deposited in the GEO

database under accession number GSE54053.

Supporting Information

S1 Figure Cbx8 induction during differentiation and generation

of a specific Cbx8 antibody. (A) The relative mRNA levels of Cbx

protein-encoding genes analyzed by qRT-PCR. Oct4 and Nanog

were included as differentiation controls. Values from untreated

cells were set to 1 and are plotted on a linear scale. (B) Schematic

representation of the human CBX8 protein. Domain structure is

according to Senthilkumar and Mishra [9]. The maximal

sequence identity to other proteins is indicated for a 50 amino

acid sliding window using the information provided by the human

protein atlas project (www.proteinatlas.org). A stretch with

minimal intraspecies identity to unrelated proteins and maximal

conservation between mouse and human (.90% identity) was

used as His-tagged antigen. (C) Scheme of the affinity purification

of the antibody. Serum from immunized rabbits is precleared with

sepharose and passed over a column containing a fusion protein of

glutathione-S-transferase (GST) and amino acids 201–360 of

CBX8 covalently cross (X)-linked to Glutathione sepharose. Anti-

CBX8 antibody is eluated with low pH and stored in PBS with

20% glycerol. (D) Total cell lysates of NTera2/D1 (NT2) cells

stably expressing shRNAs for CBX8 and controls were analyzed

by western blot using the anti-CBX8 antibody. Anti- macroH2A1

antibody was used to control loading. (E) HEK293T cells were

transiently transfected with HA-tagged CBX8 expression vector.

Immunoprecipitations were performed under regular and chro-

matin immunoprecipitation (ChIP) conditions (crosslinking, strin-

gent washing). As shown by western blot analysis using anti-HA

antibody 5 mg anti-Cbx8 antibody performed well under both

conditions. (F) Finally, we compared the performance of our anti-

Cbx8 antibody (MB) to two well described antibodies that were

kindly provided by Kristian Helin [13]. 5 mg of each antibody was

used and IgG as control. As shown on 3 target genes in ES cells

treated for three days with retinoic acid (RA), all three antibodies

enriched similarly well. (G) Specificity of the ChIP signal is shown

by the loss of signal in cells that have been infected with retroviral

shRNA vectors specific for Cbx8 or controls.

(TIF)

S2 Figure Mapped reads and peak selection. Related to Fig. 1.

(A) Details on the ChIP-seq data sets deposited in GEO. (B)

Scheme for the identification of the subset of high confidence

binding sites. We used MACS algorithm to identify peaks in the

ES.Cbx8.RA library by comparing it independently to two control

libraries, ES.IgG and ES.Cbx8.untreated. Overlapping peaks

(min. 50 bp) are considered to represent 171 high confidence

binding sites that have been analyzed further.

(TIF)

S3 Figure Gene ontologies and overlap with Ring1b and

H3K27me3. (A) Bar graphs showing up to 30 top gene ontologies

terms ordered for the –log10 adjusted p-value #0.001. Plots are

shown for all genes annotated to Cbx8 peaks and also separately

for up-, down- or not regulated genes considering a minimal fold-

change of 1.2 comparing untreated and RA-treated cells. (B) Venn

diagrams showing the overlap of Cbx8 target genes in RA-treated

cells with those of Ring1b and H3K27me3 in untreated ES cells.

(TIF)

S4 Figure Influence of shRNA-mediated knockdown of Cbx8 or

Ring1b on other PRC1 components and Cbx8 target genes.

Related to Fig. 5. (A) FLAG-epitope tagged Cbx8 is detected in

untreated ES cells binds to the same target genes as endogenous

Cbx8 in RA-treated cells. ChIP data is shown. Error bars denote

the variation of the mean of two independent experiments. (B)

Knockdown of Cbx8 does not affect global Ring1b levels. Control



www.proteinatlas.org

and Cbx8-specific shRNA expressing cells were treated with RA

for 3 days or left untreated. Lysates were analyzed by

immunoblotting. (C) Repression of Cbx8 is not compensated by

upregulation of Cbx2, Cbx4 or Cbx7. qRT-PCR analysis of

mRNA levels in the same cells as A treated with RA for three days.

Error bars denote s.d.; n = 3. (D) Control cells and cells expressing

two different Ring1b-specific shRNAs were analyzed after three

days of RA treatment. RNA was analyzed by qRT-PCR. Error

bars denote s.d.; n = 3.

(TIF)

S5 Figure Low but detectable H3K27ac is sensitive to loss of

Cbx8 in RA-treated ES cells. Related to Fig. 7. All experiments

have been performed with ES cells treated with RA for three days,

which is the time point of maximal Cbx8 recruitment (Fig. 5). (A)

The H3K27ac observed by ChIP on Cbx8 target genes Sox9 and

Tbx18 (average is plotted) is low compared to an enhancer with

well known H3K27ac enrichment (Seq1 from [49]). (B) Histone

H3K27ac levels on 4 Cbx8 target genes have been analyzed in

control cells and in cells with shRNA-mediated knockdown of

Cbx8. For each gene ChIP to input ratios of both are shown

relative to the value from control cells expressing sh Scr. Error bars

denote s.d.; n = 3; *, p,0.05. (compared to control cells).(C)

Antibodies have been titrated by ChIP to precipitate similar levels

of target gene DNA (left panel). Same amounts of antibodies were

used to reChIP from anti-Cbx8 ChIP-enriched chromatin. Error

bars represent the variation of the mean of two independent

experiments.

(TIF)

S6 Figure Neuronal differentiation of ES cells with reduced

Cbx8 expression. (A) Control and Cbx8 knockdown cells were

plated onto bacteriological Greiner Petri dishes in 15 ml of EB

medium in order to form embryoid bodies (EBs). Medium of EBs

has been changed after 2 days. Another two days later, medium

has been changed again and retinoic acid (RA) has been added.

Another two days later, medium has been changed again with

fresh medium containing RA. After 8 days of differentiation, the

EBs have been dissociated using trypsin and plated in N2B27

medium onto polyornithine and laminin-coated plates. RNA was

extracted at day 11 of the differentiation protocol and analyzed by

qRT-PCR. Data is the mean of values obtained for two

independent hairpins and is presented in respect to the value

from control cells that has been set to 1. Error bars indicate the

range of the individual values. (B) The same cells as in A were

analyzed by immunofluorescence using antibody against the

neuronal marker Tuj1.

(TIF)

S1 Table ChIP-seq peaks, annotated genes and their expression

change after RA treatment. The table contains the data that was

used to generate the Fig. 1D–F.

(XLSX)

S2 Table Proteomics data. Cbx8 and interacting proteins were

enriched by affinity purification and analyzed by mass spectrom-

etry and significantly enriched proteins were ranked according to

their abundance. The table contains the list of results from which

the table in Fig. 4B has been extracted.

(PDF)

S3 Table Oligo sequences and antibodies. The sequences of all

oligos and their position is given. For antibodies we detail sources

and amounts used for ChIP, IP and immunoblotting, respectively.

(XLSX)

Acknowledgments

We would like to express our gratitude to Lluıs Morey, Holger Richly,

Luciano Di Croce, Diego Pasini and Kristian Helin for tools and advice.

We thank Eduard Sabido, Cristina Chiva and Heinz Himmelbauer for

assistance with LC-MS/MS and Solexa sequencing.

Author Contributions

Conceived and designed the experiments: CC AP MB. Performed the

experiments: CC AP VV. Analyzed the data: CC AP RM MB.

Contributed reagents/materials/analysis tools: CC AP MB. Wrote the

paper: CC AP MB.

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a cbx8-containing polycomb complex facilitates the transition to gene activation during es cell...

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